Thermostable omega-transaminases

ABSTRACT

Thermostable omega-transaminases, particularly thermostable omega-transaminases which have a high reaction rate and which are tolerant to high concentrations of donor amine, can be used to enrich enantiomerically a mixture of chiral amines or to synthesize stereoselectively one of a pair of chiral amines in which the amino group is bound to a non-terminal, chirally substituted, carbon atom.

This application is a division of Ser. No. 11/295,696 filed Dec. 7,2005, which claims priority to provisional application Ser. No.60/634,526 filed Dec. 10, 2004. Each of these applications isincorporated by reference in its entirety herein.

FIELD OF THE INVENTION

The invention relates to omega-transaminases and their use inenantiomeric enrichment and stereoselective synthesis.

BACKGROUND OF THE INVENTION

Chiral molecules, such as single-enantiomer drugs, can be produced bybiotransformation, asymmetric synthesis using chiral catalysts orreagents, resolution or separation techniques, and the like. There is acontinuing need in the art for improved and efficient methods ofpreparing chiral molecules.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Typical reaction curves to follow the formation of product amineusing of 5 g/L and 2.5 g/L of the enzyme CNB03-03 (SEQ ID NO:12) forstereoselective synthesis at 58-60° C.

FIG. 2. Graph showing initial rates of activity of mutants CNB05-01 (SEQID NO:16) and CNB05-02 (SEQ ID NO:18).

FIGS. 3A-B. Graphs showing relative enzyme activities of CNB05-01 (SEQID NO:16) and CNB03-03 (SEQ ID NO:12). FIG. 3A, values of initial rates.FIG. 3B, rates over time.

DETAILED DESCRIPTION OF THE INVENTION

Transaminases, also called aminotransaminases, catalyze the transfer ofan amino group from a donor amine to an amine acceptor molecule.Omega-transaminases (ω-transaminases) transfer amine groups which areseparated from a carboxyl group by at least one methylene insertion. Theinvention provides thermostable omega-transaminases, particularlythermostable omega-transaminases which have a high reaction rate andwhich are tolerant to high concentrations of donor amine. Thermostableomega-transaminases of the invention can be used to enrichenantiomerically a mixture of chiral amines or to synthesizestereoselectively one of a pair of chiral amines in which the aminogroup is bound to a non-terminal, chirally substituted, carbon atom.Methods of the invention provide cost-effective and specific means forproducing chiral molecules.

Thermostable Omega-Transaminases

The amino acid sequence of a wild-type omega-transaminase fromArthrobacter citreus is shown in SEQ ID NO:2. Thermostable omegatransaminases of the invention comprise an amino acid sequence whichdiffers from the amino acid sequence shown in SEQ ID NO:2 at each ofpositions 242, 245, 255, and 268 with one or more additional amino acidsubstitutions at positions 46, 48, 60, 164, 185, 186, 195, 197, 205,252, 323, 409, 424, and 436.

For example, some thermostable transaminases have an additional aminoacid substitution at position 46 (e.g., SEQ ID NOS:12, 14, 16, and 18)or at position 185 (e.g., SEQ ID NOS:16 and 18). Others have additionalamino acid substitutions at positions 48, 195, and 197 (e.g., SEQ IDNOS:8, 10, 12, 14, 16, and 18); positions 46, 48, 195, and 197 (e.g.,SEQ ID NOS:16 and 18); positions 60, 164, 186, 252, 409, and 426 (e.g.,SEQ ID NOS:6, 8, 10, 12, 16, and 18); positions 48, 60, 164, 186, 195,197, 252, 409, and 436 (e.g., SEQ ID NOS:8 and 12); positions 48, 60,164, 186, 195, 197, 252, 409, 424, and 436 (SEQ ID NOS: 8, 10, and 12);or positions 46, 48, 60, 164, 185, 186, 195, 197, 409, 424, and 436(e.g., SEQ ID NOS:16 and 18).

Certain thermostable omega transaminases of the invention comprise anamino acid sequence which differs from the amino acid sequence shown inSEQ ID NO:2 at each of positions 60, 164, 186, 242, 245, 252, 255, 268,409, and 436; in these transaminases, one or more substitutions also canbe made at positions 46, 48, 195, 197, and 424.

Other thermostable omega transaminases of the invention comprise anamino acid sequence which differs from the amino acid sequence shown inSEQ ID NO:2 at each of positions 46, 48, 195, 197, 242, 245, and 255; inthese transaminases, one or more substitutions also can be made atpositions 60, 164, 185, 186, 205, 252, 323, 409, 424, and 436.

Preferred substitutions are provided in Table 1 and include all possiblecombinations of the possible substitutions shown. The most preferredsubstitutions are indicated in bold and underlined. TABLE 1 Preferredamino acid substitutions with respect to SEQ ID NO: 2. amino acidposition no. wild-type thermostable omega-transaminase 46 Met Gly, Ser,Thr , Cys, Tyr, Asp, Glu, Ala, Val, Leu, Ile, Pro, Phe, and Trp 48 AspGly , Ser, Thr, Cys, Tyr, and Glu 60 Tyr Gly, Ser, Thr, Cys , Asp, andGlu 164 Tyr Ala, Val, Leu, Ile, Pro, Phe , Trp, and Met 185 Tyr Gly,Ser, Thr, Cys , Asp, and Glu 186 Asp Gly, Ser , Thr, Cys, Tyr, and Glu195 Pro Gly, Ser , Thr, Cys, Tyr, Asp, Glu, Ala, Val, Leu, Ile, Phe,Trp, and Met 197 Met Gly, Ser, Thr , Cys, Tyr, Asp, Glu, Ala, Val, Leu,Ile, Pro, Phe, and Trp 205 Cys Tyr , Gly, Ser, Thr, Asp, and Glu 242 AlaVal , Leu, Ile, Pro, Phe, Trp, and Met 245 Ala Gly, Ser, Thr , Cys, Tyr,Asp, and Glu 252 Ile Ala, Val , Leu, Pro, Phe, Trp, and Met 255 Phe Ala,Val, Leu, Ile , Pro, Trp, and Met 268 Asp Gly, Ser , Thr, Cys, Tyr, andGlu 323 His Tyr , Gly, Ser, Thr, Cys, Asp, and Glu 409 Thr Lys, Arg ,and His 424 Lys Glu , Asp, Arg, and His 436 Val Ala , Leu, Ile, Pro,Phe, Trp, and Met

Omega-transaminases with the substitutions described in Table 1 arethermostable. “Thermostable” means that the enzyme is active (able tocarry out the reaction, either stereoselective synthesis or enantiomericenrichment, as indicated by the continued formation of product) andstable (active for at least 10 hours) up to at least 40° C., preferablyup to at least 50-60° C. (e.g., at least 50, 51, 52, 53, 54, 55, 56, 57,58, 59, or 60° C.), even more preferably up to at least 55-63° C. (e.g.,at least 55, 56, 57, 58, 59, 60, 61, 62, or 63° C.). Activity ismeasured by the monitoring the formation of product as a function oftime in a reaction. Typical stereoselective synthesis and enantiomericenrichment reactions are described in Example 1.

Such thermostable omega-transaminases also have a greater tolerance tohigh donor amine concentrations than the wild-type enzyme (i.e., theyare active and stable) in the presence of high concentration of amine.Preferred thermostable omega-transaminases are active and stable for atleast 10 hours in the presence of >500 mM amine. Omega-transaminaseswith optional additional substitutions at positions 46, 48, 195, 197,and 424 also have higher reaction rates than the wild-type enzyme. Thesehigher reaction rates typically are 1.2 to 3 times higher than thereaction rate of the wild-type enzyme as measured by the rate offormation of product (see Example 1).

The amino acid sequences of several thermostable omega-transaminases areshown in SEQ ID NOS:6 (“Chir9867”), 8 (“CNB03-01”), 10 (“CNB03-02”), 12(“CNB03-03”), 14 (“CNB04-01”), 16 (“CNB05-01”), and 18 (“CNB05-02”) andin Table 2, below. In Table 2, amino acid substitutions with respect tothe wild-type sequence are shown in bold. Thermostableomega-transaminases of the invention do not have the amino acid sequenceshown in SEQ ID NO:4 and encoded by the nucleotide sequence shown in SEQID NO:3; SEQ ID NO:4 is the amino acid sequence of an omega-transaminasewhich is tolerant to high donor amine concentrations but which is notthermostable. TABLE 2 Amino acid substitutions in thermostableomega-transaminases. amino wild-type CNB04-01 CNB05-01 CNB05-02 acid(SEQ ID NO: Chir9867 CNB03-01 CNB03-02 CNB03-03 (SEQ ID (SEQ ID (SEQ IDposition 2) (SEQ ID NO: 6) (SEQ ID NO: 8) (SEQ ID NO: 10) (SEQ ID NO:12) NO: 14) NO: 16) NO: 18) 46 Met Met Met Met Thr Thr Thr Thr 48 AspAsp Gly Gly Gly Gly Gly Gly 60 Tyr Cys Cys Cys Cys Tyr Cys Cys 164 TyrPhe Phe Phe Phe Tyr Phe Phe 185 Tyr Tyr Tyr Tyr Tyr Tyr Cys Cys 186 AspSer Ser Ser Ser Asp Ser Ser 195 Pro Pro Ser Ser Ser Ser Ser Ser 197 MetMet Thr Thr Thr Thr Thr Thr 205 Cys Cys Cys Cys Cys Cys Tyr Cys 242 AlaVal Val Val Val Val Val Val 245 Ala Thr Thr Thr Thr Thr Thr Thr 252 IleVal Val Val Val Val Val Ile 255 Phe Ile Ile Ile Ile Ile Ile Ile 268 AspSer Ser Ser Ser Ser Ser Ser 323 His His His His His His His Tyr 409 ThrArg Arg Arg Arg Thr Arg Arg 424 Lys Glu Glu Lys Glu Lys Glu Glu 436 ValAla Ala Ala Ala Val Ala Ala

Preparation of Thermostable Omega-Transaminases

Thermostable omega-transaminases of the invention can be produced by anysuitable means known in the art (e.g., recombinantly, for example in ahost cell, or by chemical synthesis). Preferably the enzymes areproduced using high cell density fermentation with recombinant E. coli.

Recombinant Production of Thermostable Omega-Transaminases Nucleic AcidMolecules

The sequence listing provides coding sequences for preferredthermostable omega-transaminases of the invention (SEQ ID NOS:5, 7, 9,11, 13, 15, and 17 encode SEQ ID NOS:6, 8, 10, 12, 14, 16, and 18,respectively). Any nucleotide sequence which encodes a particularthermostable omega-transaminase, however, can be used to produce thatenzyme recombinantly. For example, sequences encoding a thermostableomega-transaminase can be synthesized, in whole or in part, usingchemical methods well known in the art (see Caruthers et al., Nucl.Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp.Ser. 225-232, 1980). Alternatively, nucleic acid molecules encoding thewild-type omega-transaminase of SEQ ID NO:2 (e.g., SEQ ID NO:1) can bemodified to encode a thermostable omega-transaminase of the invention.If desired, such molecules can be isolated from Arthrobacter citreusbacteria using standard nucleic acid purification techniques or can besynthesized using an amplification technique, such as the polymerasechain reaction (PCR), or by using an automatic synthesizer. Methods forisolating nucleic acids are routine and are known in the art. Any suchtechnique for obtaining a nucleic acid molecules can be used to obtain anucleic acid molecule which encodes the wild-type omega-transaminase.

cDNA molecules encoding thermostable omega-transaminases of theinvention can be made with standard molecular biology techniques, usingmRNA as a template. cDNA molecules can thereafter be replicated usingmolecular biology techniques well known in the art. An amplificationtechnique, such as PCR, can be used to obtain additional copies ofpolynucleotides of the invention.

If desired, the nucleotide sequences disclosed herein can be engineeredusing methods generally known in the art to alter coding sequences forthermostable omega-transaminases of the invention for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing, and/or expression of the polypeptide or mRNAproduct. Sequence modifications, such as the addition of a purificationtag sequence or codon optimization, can be used to facilitateexpression. These methods are well known in the art and are furtherdescribed, for example, in PCT/US04/024868.

Expression Vectors

A nucleic acid molecule which encodes a thermostable omega-transaminaseof the invention can be inserted into an expression vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining sequences encoding enzymes of the invention and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques as well as synthetictechniques.

Host Cells

A host cell can be prokaryotic or eukaryotic. Useful host cells includeArthrobacter citreus itself, E. coli, Bacillus subtilis, Vibriocholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica,Neisseria cinerea, Mycobacteria (e.g., M. tuberculosis), yeasts, etc.Many types of host cells are available, for example, from the AmericanType Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va.20110-2209) and can be chosen to ensure the correct modification andprocessing of a foreign protein. See, e.g., WO 01/98340.

Expression constructs can be introduced into host cells usingwell-established techniques. Such techniques include, but are notlimited to, transferrin-polycation-mediated DNA transfer, transfectionwith naked or encapsulated nucleic acids, liposome-mediated cellularfusion, intracellular transportation of DNA-coated latex beads,protoplast fusion, viral infection, electroporation, “gene gun,” andDEAE- or calcium phosphate-mediated transfection.

Host cells transformed with expression vectors can be cultured underconditions suitable for the expression and recovery of the enzyme fromthe cell culture. The enzyme produced by a transformed cell can besecreted or contained intracellularly depending on the nucleotidesequence and/or the expression vector used.

Purification of Thermostable Omega-Transaminases

A recombinantly produced thermostable omega-transaminase can be isolatedfrom a host cell engineered to produce the enzyme. A purifiedthermostable omega-transaminase is separated from other components inthe cell, such as proteins, carbohydrates, or lipids, using methodswell-known in the art. Such methods include, but are not limited to,size exclusion chromatography, ammonium sulfate fractionation, ionexchange chromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified thermostableomega-transaminase is at least 80% pure; preferably, the preparationsare 90%, 95%, or 99% pure. Purity of the preparations can be assessed byany means known in the art, such as SDS-polyacrylamide gelelectrophoresis.

Chemical Synthesis of Thermostable Omega-Transaminases

Thermostable omega-transaminases of the invention can be synthesized,for example, using solid-phase techniques. See, e.g., Merrifield, J. Am.Chem. Soc. 85, 2149-54, 1963; Roberge et al., Science 269, 202-04, 1995.Protein synthesis can be performed using manual techniques or byautomation. Automated synthesis can be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally,fragments of a thermostable omega-transferase can be synthesizedseparately and combined using chemical methods to produce a full-lengthmolecule.

Methods of Preparing Chiral Molecules

The invention provides methods of using thermostable omega-transaminasesto prepare chiral molecules, either by enantiomerically enriching amixture of chiral amines or by stereoselectively synthesizing one of apair of chiral amines.

The enzymatic equilibrium reaction in which omega-transaminasesparticipate is illustrated below:

in which each of R¹ and R², when taken independently, is an alkyl oraryl group which is unsubstituted or substituted with one or moreenzymatically non-inhibiting groups and R¹ is different from R² instructure or chirality, or R¹ and R², taken together, are a hydrocarbonchain of 4 or more carbon atoms containing a center of chirality.

Amino Acceptors

An “amino acceptor” is a carbonyl compound which accepts an amino groupfrom a donor amine. Amino acceptors include ketocarboxylic acids andalkanones. Typical ketocarboxylic acids are α-keto carboxylic acids suchas glyoxalic acid, pyruvic acid, oxaloacetic acid, and the like, as wellas salts of these acids. Amino acceptors also include or substanceswhich are converted to an amino acceptor by other enzymes or whole cellprocesses, such as fumaric acid (which can be converted to oxaloaceticacid), glucose (which can be converted to pyruvate), lactate, maleicacid, etc.

Amino Donors

An “amino donor” is an amino compound which donates an amino group tothe amino acceptor, thereby becoming a carbonyl species. Typical aminodonors include the nonchiral amino acid glycine and chiral amino acidshaving the S-configuration such as L-alanine or L-aspartic acid; aminodonors, however, need not be amino acids. For example, amines such asS-2-aminobutane, propyl amine, benzyl amine, etc. also can be used asamino donors.

Chiral Amines

Chiral amines have the formula:

in which each of R¹ and R² are as defined above. The compounds ofFormulas IA and IB are enantiomers (or diastereomers if either R¹ or R²contains a second chiral center) and are chiral because R¹ is differentin structure or chirality from R².

In chiral amines of Formulas IA and IB, the amino group is a primaryamine and is bound to a secondary carbon atom, i.e., a carbon atomcarrying one hydrogen atom and two substituents which are other thanhydrogen (R¹ and R²). In addition, while R¹ and R² are selected from thesame type of structures, these groups must render the molecule chiral;e.g., R¹ will be different from R² in structure or chirality or R¹ andR² when taken together are a chiral group. Generally, when takenindependently, R¹ and R² will be alkyl, aralkyl, or aryl groups,preferably a straight or branched alkyl group of from 1 to 6 carbonatoms, a straight or branched phenyl-alkyl group of from 7 to 12 carbonatoms, or a phenyl or naphthyl group. Examples include methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, phenyl, benzyl,phenethyl, 2-phenylpropyl, etc.

Each R¹ and R² group optionally can be substituted with one or moregroups, provided the groups do not significantly affect or compete withthe action of the omega-transaminase. This can be readily determined bya simple inhibition assay. If inhibition is detected, it often can beminimized by conducting the reaction at lower concentrations of thatreactant. Typical substituents without limitation include halo such aschloro, fluoro, bromo and iodo, hydroxy, lower alkyl, lower alkoxy,lower alkylthio, cycloalkyl, carbamoyl, mono- and di-(lower alkyl)substituted carbamoyl, trifluoromethyl, phenyl, nitro, amino, mono- anddi-(lower alkyl) substituted amino, alkylsulfonyl, arylsulfonyl,alkylcarboxamido, arylcarboxamido, etc. Typical groups when R¹ and R²are taken together are 2-methylbutane-1,4-diyl, pentane-1,4-diyl,hexane-1,4-diyl, hexane-1,5-diyl, and 2-methylpentane-1,5-diyl.

Typical amines for which the present process is suitable include withoutlimitation 2-aminobutane, 2-amino-1butanol, 1-amino-1-phenylethane,1-amino-1-(2-methoxy-5-fluorophenyl)ethane, 1-amino-1-phenylpropane,1-amino-1-(4hydroxyphenyl)propane, 1-amino-1-(4-bromophenyl)propane,1-amino-1-(4-nitrophenyl)propane, 1-phenyl-2-aminopro-pane,1-(3-trifluoromethylphenyl)-2-aminopropane, 2-aminopropanol,1-amino-1-phenylbutane, 1-phenyl-2-aminobutane,1-(2,5-di-methoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane,1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-2-methylcyclopentane,1-amino-3-methylcyclopentane, 1-amino-2-methylcyclohexane,1-amino-1-(2-naphthyl)ethane, 3-methylcyclopentylamine,2-methylcyclopentylamine, 2-ethylcyclopentylamine,2-methylcyclohexylamine, and 3-methylcyclohexylamine, 1-aminotetralin,2-aminotetralin, 2-amino-5-methoxytetralin, and 1-aminoindan.

Thermostable Omega-Transaminases

Any of the thermostable omega-transaminases described above can be usedto produce chiral compounds. A thermostable omega-transaminase can beused in free form (e.g., as a purified enzyme or in a cell-freeextract). The enzyme optionally can be immobilized on a suitable supportor matrix, such as cross-linked dextran or agarose, silica, polyamide,or cellulose. The enzyme also can be encapsulated in polyacrylamide,alginates, fibers, or the like. Methods for such immobilization aredescribed in the literature (see, for example, Methods in Enzymology 44,1976). The latter embodiment is particularly useful because, forexample, once the immobilized enzyme is prepared one can simply feed theamino acceptor and a mixture of the chiral amines over the immobilizedenzyme in order to effect the desired enrichment, and then remove theformed ketone. See U.S. Pat. Nos. 4,950,606; 5,300,437; and 5,346,828.

Enantiomeric Enrichment of a Chiral Amine

In some embodiments, thermostable omega-transaminases of the inventionare used in methods for enantiomeric enrichment of a mixture of chiralamines. “Enantiomeric enrichment” is an increase in the amount of onechiral form compared to the other. This enrichment can involve (i) adecrease in the amount of one chiral form compared with the other, (ii)an increase in the amount of one chiral form compared with the other, or(iii) a decrease in the amount of one chiral form and an increase in theamount of the other chiral form. Enantiomeric enrichment typically isexpressed as “enantiomer excess,” or “ee,” according to the followingexpression: ${ee} = {\frac{E^{1} - E^{2}}{E_{1} + E^{2}} \times 100}$in which E¹ is the amount of the first chiral form of the amine and E²is the amount of the second chiral form of the same amine. For example,if the initial ratio of the two chiral forms is 50:50 and anenantiomeric enrichment sufficient to produce a final ratio of 50:30 isachieved, the ee with respect to the first chiral form is 25%, whereasif the final ratio is 70:30, the ee with respect to the first chiralform is 40%. Typically with methods of the present invention, an ee of90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or greater can be achieved.Enantiomeric enrichment can be determined by any means known in the art.For example, the ee of a given product can be determined by reactionwith (−) α-(tri-fluoromethylphenyl)methoxyacetyl chloride (Gal, J.Pharm. Sci. 66, 169, 1977; Mosher et al., J. Org. Chem. 34, 25430, 1969)followed by capillary gas chromatography of the derivatized product on aChrompack fused silica column.

Broadly, methods of enantiomeric enrichment involve subjecting a mixtureof chiral amines to the action of a thermostable omega-transaminase ofthe invention in the presence of an amino acceptor.

in which R¹ and R² are as defined above and, in Formula III, either R³is R¹ while R⁴ is R² or R³ is R² while R⁴ is R¹.

In general, the enzymatic process operates on only one chiral form, oroperates on one chiral form to a far greater extent than the other.Because the reaction is an equilibrium, either the forward or reversereactions can be favored by adding additional starting materials orremoving reaction products. Thus, in an enantiomeric enrichmentreaction, additional quantities of the amino acceptor can be added (upto saturation) and/or the ketone formed can be continuously removed fromthe reaction mixture. Conversely when one stereoselectively synthesizesone chiral form of an amine, as described below, additional ketone canbe added (up to saturation) and/or the amine formed can be removed.

When the undesired chiral form of the amine is converted to the ketoneand the desired chiral form is not, the latter can be readily isolatedby conventional techniques. A partial separation can be effected byacidification, extraction with a hydrocarbon such as heptane to removethe ketone, rendering the aqueous phase basic, and re-extraction with ahydrocarbon such as heptane. When, on the other hand, both chiral formsof the amine are desired, the form which is converted to the ketone canbe removed from the reaction mixture (or from the aqueous phase in a twophase mixture) and independently subjected to the action of anomega-transaminase in the presence of a amino donor to generate the samechiral form which was initially converted to the ketone.

Stereoselective Synthesis of a Chiral Form

Thermostable omega-transaminases of the invention also can be used inmethods of stereoselective synthesis, i.e., to preferentially synthesizeone chiral form of an amine of formula IA or IB in an amountsubstantially greater than the other. These methods typically involvesubjecting a ketone of the formula:

in which R¹ and R² are as defined above to the action of a thermostableomega-transaminase of the invention in the presence of an amino donoruntil a substantial amount of one of the chiral amines is formed.“Substantially greater” as used herein refers to a percentage of thecombined amount of both chiral forms of at least about 51% (e.g., atleast 51, 55, 60, 65, 70 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,or 99%).

All patents, patent applications, and references cited in thisdisclosure are expressly incorporated herein by reference. The abovedisclosure generally describes the present invention. A more completeunderstanding can be obtained by reference to the following specificexamples, which are provided for purposes of illustration only and arenot intended to limit the scope of the invention.

EXAMPLE 1

Stereoselective Synthesis and Enantiomeric Enrichment Reactions

To assess activity of a thermostable omega-transaminase in astereoselective synthesis reaction, 108.8 g potassium phosphate is addedto water in a reactor. One liter of concentrated HCl is added, and thenisopropylamine is added. The pH is adjusted to 7-7.4, and the volume isadjusted to 8 L (i.e., 100 mM potassium phosphate and 1.0 Misopropylamine). Pyridoxal-5-phosphate is added to a final concentrationof 2 mM, followed by 1-5 g of spray-dried crude thermostableomega-transaminase. Preferably the reaction takes place in the absenceor near absence of oxygen, so nitrogen flow is started to purge thereaction mixture of oxygen. The enzyme is mixed for one hour under anitrogen atmosphere, pH adjusted to 7-7.2, and the reaction is startedby adding ketone 7-methoxy-2-tetralone to a final concentration of 100mM. Samples are taken at regular intervals. Stability and activity aremeasured by monitoring the formation of amine product for at least 10hours. FIG. 1 shows the typical progress of a synthesis reaction usingthe enzyme CNB03-03.

To assess activity of a thermostable omega-transaminase in anenantiomeric enrichment reaction, racemic methylbenzylamine is used asthe amine donor, and sodium pyruvate is used as the amine acceptor; therest of the components are the same.

EXAMPLE 2

Identification of Thermostable Transaminases

“CHIR-9867” (SEQ ID NO:6) is a mutant, thermostable transaminase.Mutants of CHIR-9867 were generated using error-prone PCR (Leung et al.,J. Methods Cell. Mol. Biol. 1, 11-15, 1989) and screened for theiractivity at 60-68° C. compared to CHIR-9867 on a plate screen for thesynthesis of S-7-Methoxy-2-aminotetralin (S7MAT). The plate screen isperformed by subjecting the mutants to a reaction whereby an amine isconverted into a ketone that becomes colored in the presence of oxygen.The rate at which the color is formed is used as an indication of theactivity of the enzyme.

Thirty-eight potential candidates were identified from a primary platescreen. All identified mutants were further screened on plates, andthirteen mutants were identified as potential improved mutants. These 38mutants appeared to have a better rate than the wild-type enzyme. Thesethirteen mutants were screened on 3 mL scale (reaction volume was 3 mL)at ≈65° C. The three best mutants were identified and named CNB03-01,CNB03-02, CNB03-03. These three identified mutants were further screenedon 40 mL scale under a nitrogen atmosphere at different temperatures andenzyme concentrations to select the mutant with the best reaction rate.The reaction rate of CHIR-9867 was used as a control. The resultsobtained from the 40 mL scale screening are shown in Table 3. TABLE 3Initial Quantity Rate Enzyme # (g/L) Reaction Conditions (mM/hr) CNB03-01 15 160 mM ketone, 650 mM 68 donor amine, 60-62° C. CNB 03-02 15160 mM ketone, 58 650 mM donor amine, 60-62° C. CNB 03-03 15 160 mMketone, 71 650 mM donor amine, 60-62° C. CHIR-9867 15 160 mM ketone, 28650 mM donor amine, 60-62° C.

All mutants showed high selectivity and the amine was synthesized withan ee >99%. Chiral purity was measured using HPLC with a chiral column.

CNB 03-01, CNB 03-02, and CNB 03-03 were sequenced. Their amino acidsequences are shown in SEQ ID NOS:8, 10, and 12, respectively. Asexpected, all of these mutant transaminases were altered in amino acidsequence compared to the amino acid sequence of CHIR-9867 (SEQ ID NO:6)from which they were generated. The changes in amino acid sequencesbetween CNB 03-01, CNB 03-02, CNB 03-03, and CHIR-9867 are shown inTable 2.

EXAMPLE 3

Summary of HPLC Analytical Methods Used for Chemical and Chiral AnalysisHPLC Method for Chemical Analysis Column: Waters, Nova-Pak Phenyl RCM0.8 cm × 10 cm × 4 μm Mobile Phase: 25/75 (v/v-premixed)isopropanol/water containing 0.15% H₃PO₄ and 10 mM sodiumoctanesulfonate Flow Rate: 1.5 ml/min Injection Volume: 10 μLTemperature: Ambient Run Time: 20 min Detection: UV @ 254 nm TypicalRetention times: ketone, 9-10 min; amine 13-14 min

HPLC Method for Chiral Analysis Column: Diacel Crownpak CR (+) 15 cm ×0.40 cm × 5 μm Mobile Phase: 15/85 (v/v-premixed) methanol/1% HClO₄ inwater. Flow Rate: 1.25 ml/min Injection Volume: 15 μL Temperature: 40°C. Run Time: 50 min Detection: UV @ 220 nm Typical Retention R-amine20-21 minutes; S-amine, 24-25 min. Times:

EXAMPLE 4

Generation of a New Enzymes, CNB04-01, with Extended Thermostability

“CNB03-03” (SEQ ID NO:10) is a mutant, thermostable transaminase.Mutants of CNB03-03 were generated using error-prone PCR (Leung et al.,J. Methods Cell. Mol. Biol. 1, 11-15, 1989) and screened for theiractivity after more than 24 hours at 60-68° C. compared to CNB03-03 on aplate screen for the synthesis of S-7-Methoxy-2-aminotetralin (S7MAT).The plate screen was performed by subjecting the mutants to a reactionwhereby an amine is converted into a ketone that becomes colored in thepresence of oxygen. The rate at which the color is formed was used as anindication of the activity of the enzyme.

Thermostability of CNB04-01 was tested as follows: enzyme was kept at50° C. in a solution of 100 mM calcium chloride, 100 mM sodium acetate,1 mM pyridoxal-5-phosphate at pH 8.5 (0.1 gram enzyme dissolved in 10 mlof solution). Periodically, 1 ml samples were withdrawn and added to 20ml of reaction mixture pH 8.5 and incubated at 50° C. The change on(S)-aminotransferase activity as a function of time was measured.Results showed that the activity of the enzyme remained relativelystable for over a period of 150 hours with the initial rate at time zeroof ˜36 mM/hour and an initial rate of ˜25 mM/hour after 150 hours.

EXAMPLE 5

Initial Rates of Activity of CNB05-01 (SEQ ID NO:16) and CNB05-02 (SEQID NO:18)

The initial rates of activity of the thermostable transaminases CNB05-01(SEQ ID NO:16) and CNB05-02 (SEQ ID NO:18) were tested under thefollowing reaction conditions: V=50 ml [2 mM PLP, 0.5 IPA, 200 mM sodiumacetate], Enzyme: 5 g/l; (pH)0=7, ketone=130 mM, 55° C.

The results are shown in FIG. 2. From FIG. 2 it is clear that theinitial rate of CNB05-01 and CNB05-02 is greater than CNB03-03. Theinitial rate of CNB05-01 was better than all the enzymes tested and was0.39 mM/minute under the conditions tested (CNB03-03 had an initial rateof 0.21 mM/minutes).

EXAMPLE 6

Comparison of Enzymatic Activity of CNB05-01 (SEQ ID NO:16) and CNB03-03(SEQ ID NO:12)

Enzymatic activity of the thermostable transaminases CNB05-01 (SEQ IDNO:16) and CNB03-03 (SEQ ID NO:12) were tested under the followingconditions: 55° C., pH 7 (0-5 hr), 100 ml/min N2, 0.75 M IPA, 2 mM PLP,200 mM sodium acetate, 2 ml ketone (130 mM), V=100 ml, nitrogen flowsurface 100 ml/min.

The results are shown in FIGS. 3A and 3B. FIG. 3A demonstratesreproducibility of the results described in this example with theinitial rate of CNB05-01 at 0.41 mM/minute and CNB03-03 at 0.25mM/minute. FIG. 3B shows the results obtained during the course of thereaction with CNB05-01 achieving a higher conversion in a shorter periodof time by virtue of having a higher initial rate.

1. An isolated thermostable omega transaminase which comprises the aminoacid sequence SEQ ID NO:16.
 2. An isolated polynucleotide which encodesthe amino acid sequence SEQ ID NO:16.
 3. The isolated polynucleotide ofclaim 2 which comprises the nucleotide sequence SEQ ID NO:15.
 4. Theisolated polynucleotide of claim 2 which is an expression construct. 5.A method for enantiomeric enrichment of a mixture of two enantiomericchiral amines comprising contacting: (a) a mixture comprising a firstchiral amine and a second chiral amine which is an enantiomer of thefirst chiral amine, wherein the first chiral amine has the structuralformula:

and wherein the second chiral amine has the structural formula:

 wherein each of R¹ and R², when taken independently, is alkyl,arylalkyl, or aryl and may be unsubstituted or substituted with one ormore enzymatically non-inhibiting groups, and wherein R¹ and R² renderthe molecule chiral; (b) a thermostable omega transaminase whichcomprises the amino acid sequence SEQ ID NO:16; and (c) an aminoacceptor for a time sufficient to convert the first chiral amine to aketone such that the amount of the second chiral amine in the mixture isat least about 90% relative to the amount of the first chiral amine. 6.The method of claim 5 wherein the contact is maintained at least untilthe amount of the second chiral amine in the mixture is at least about99% relative to the amount of the first chiral amine.
 7. The method ofclaim 5 wherein the one or more enzymatically non-inhibiting groups areselected from the group consisting of halogen, hydroxy, lower alkyl,lower alkoxy, lower alkylthio, cycloalkyl, carbamoyl, mono-(loweralkyl)-substituted carbamoyl, di-(lower alkyl)-substituted carbamoyl,trifluoromethyl, phenyl, nitro, amino, mono-(lower alkyl) substitutedamino, di-(lower alkyl) substituted amino, alkylsulfonyl, arylsulfonyl,alkylcarboxamido, arylcarboxamido, 2-methylbutane-1,4-diyl,pentane-1,4-diyl, hexane-1,4-diyl, hexane-1,5-diyl, and2-methylpentane-1,5-diyl.
 8. The method of claim 5 wherein the aminoacceptor is a ketocarboxylic acid.
 9. The method of claim 8 wherein theketocarboxylic acid is selected from the group consisting of glyoxalicacid, pyruvic acid, oxaloacetic acid, and salts thereof.
 10. The methodof claim 5 wherein the amino acceptor is selected from the groupconsisting of oxaloacetic acid and pyruvic acid.
 11. A method forstereoselective synthesis of one of two chiral forms of an aminecomprising contacting: (a) a ketone having the structural formula:

 wherein each of R¹ and R², when taken independently, is alkyl,arylalkyl, or aryl and may be unsubstituted or substituted with one ormore enzymatically non-inhibiting groups; (b) a thermostable omegatransaminase which comprises the amino acid sequence SEQ ID NO:16; and(c) an amino donor for a time sufficient to form a substantially greateramount of a first chiral form of the amine than the amount of a secondchiral form of the amine.
 12. The method of claim 11 wherein the one ormore enzymatically non-inhibiting groups are selected from the groupconsisting of halogen, hydroxy, lower alkyl, lower alkoxy, loweralkylthio, cycloalkyl, carbamoyl, mono-(lower alkyl)-substitutedcarbamoyl, di-(lower alkyl)-substituted carbamoyl, trifluoromethyl,phenyl, nitro, amino, mono-(lower alkyl) substituted amino, di-(loweralkyl) substituted amino, alkylsulfonyl, arylsulfonyl, alkylcarboxamido,arylcarboxamido, 2-methylbutane-1,4-diyl, pentane-1,4-diyl,hexane-1,4-diyl, hexane-1,5-diyl, and 2-methylpentane-1,5-diyl.
 13. Themethod of claim 11 wherein the amino donor is selected from the groupconsisting of glycine, L-alanine, L-aspartic acid, S-2-aminobutane,propyl amine, and benzyl amine.